Abstract: Opiate abuse and HIV-1 have been described as two linked global health crises. Despite the advent of anti- retroviral therapy, there is increased prevalence of neurologic and cognitive deficits in HIV-1 infected individuals. In our previous study using the well-established rhesus macaque (RM) model infected with CCR5-utilizing SIV R71/17E virus, we demonstrated robust microglial activation and migration in morphine-dependent, SIV-infected RMs compared with SIV-infected RMs without opiate dependence. The goal of the current proposal is to design and develop an extracellular vesicle (EV)-based strategy of in vivo anti-microRNA (miR) delivery as a means to ameliorate morphine & HIV protein (Tat)-induced microglial activation & migration in the brain. This proposal is focused specifically on the generation and optimization of engineered EVs containing CNS targeted peptides (to facilitate their localization in the brain) that will be loaded with key anti-miRs (see below) with the ultimate goal of testing their efficacy in blocking microglial activation & migration mediated by morphine and HIV Tat, respectively, in a rodent model of HAND. MicroRNA (miR)-mediated regulation of disease pathogenesis is an evolving research area that has implications for identification of potential therapeutic targets for various neurodegenerative disorders for which currently there exists no cure. EVs are becoming well recognized as cell- cell communication conduits that deliver the cargo containing miRNA, mRNA and proteins to the neighboring/distant cells. Previous findings by others and us demonstrate that exposure of astrocytes to morphine & HIV protein Tat resulted in upregulated expression & release of miRs-138 & -9 in the EVs, which, upon uptake by the microglia, led to the activation & migration of microglia, respectively. In addition, we have also validated these findings in vivo, wherein microinjection of miR-138-loaded EVs into the mouse brain resulted in significant activation of microglia. Based on these findings we hypothesize that intranasal delivery of engineered brain-targeting EVs loaded with anti-miRs (-138 & -9) to morphine administered, Tat-inducible mice will lead to uptake of anti-miRs by microglia, thereby resulting in abrogation of both microglial activation & migration. This hypothesis will be tested via two specific aims. Specific Aim 1: To design, engineer and optimize EVs loaded with brain-targeting peptide and anti-miRNAs in vitro for their ability to block morphine and HIV Tat- mediated microglial activation and migration, respectively. Specific Aim 2: To test the in vivo efficacy of optimized EVs (from SA1) in ameliorating morphine & HIV Tat induced microglial activation & migration, respectively. Our long-term goal is to develop EV-based RNA drug delivery in vivo as a therapeutic for use in future preclinical trials on the treatment of HAND in opiate abusers.